1. Field of the Invention
A bienzyme electrode contains a peroxidase and an oxidase. It is generally useful in bioelectrochemical assays, for example for determining the substrates of ethanol oxidase, choline oxidase, cholesterol oxidase or a D-amino-acid oxidase.
2. Brief Description of the Related Art
Enzyme electrodes have gained considerable importance with regard to determinations of metabolites in medicine, biotechnology, food technology and many other areas of science and technology; Schmide, R. D. et al. (1988). Biosensors and "Bioelectronics". In Biotechnology (ed. H. -J. Rehm, G. Reed) VCH Verlagsgesellschaft, Wein, 6b, 317. Bienzyme systems which contain peroxidases and oxidases have been used for the manufacture of highly selective amperometric enzyme electrodes; Kulys, J. J. et al. (1981). Biolectrochem. Bioenerg., 8, 81-88. Ferrocyanide was used as soluble mediator. These electrodes were not suitable for use in vivo or for monitoring bioreactors. Other systems which contained organometallic compounds and peroxidase/oxidase did not need a mediator but the efficiency of the peroxidase activity was low, and the electrodes exhibited a delayed response and insufficient stability.
We have proposed the use of a peroxidase from the fungus Arthromyces ramosus; Shinmen, Y. et al. (1986). Agric. Biol. Chem., 50, 247-249; or of chemically modified electrodes; Cenas, N. K. et al. (1981). Biocelectrochem. Bionerg., 8, 103-113, in order to increase the efficiency of the peroxidase activity and produce a bienzyme electrode without mediator. To our knowledge no fungal peroxidase has previously been used to manufacture an electrochemical system even though, depending on the substrate used, it has shown a catalytic activity which is 2.9 to 540 times higher than horseradish peroxidase; Brochure: neue Peroxidase aus dem Pilz Arthromyces ramosus (Novel Peroxidase from the fungus Arthromyces ramosus). Suntory Ltd. Institute for Fundamental Research Mishima-gun, Osaka 618 Japan.
Chemical modifications have been widely used in the manufacture of enzyme electrodes; Aston, W. J. (1987), Biosensors Fundamentals and Application (Ed. A. P. F. Turner, I. Karube, G. S. Wilson), Oxford University Press, Oxford, New York, Tokyo, 276-291. The first chemically modified electrode contained tetracyanoquinodimethane and the potassium salt thereof Cenase et al., supra. Unfortunately some oxidases, such as, for example, choline oxidase, cholesterol oxidase, amino-acid oxidase and D-amino-acid oxidase do not react with modifying substances; Davis, G. (1977). Biosensors Fundamentals and Application (Ed. A. P. F. Turner, I. Karube, G. S. Wilson), Oxford University Press, Oxford, New York, Tokyo, 247-257 so that it is impossible to construct an electrode which simply contains only oxidase.